METHODS:

Caco-2 cells that were either nontransfected or stably transfected with human TNF receptor 1 (TNFR1) or TNFR2 and mouse colonocytes were used for physiologic, morphologic, and biochemical analyses.

RESULTS:

Colitis induced in vivo by adoptive transfer of CD4(+)CD45RB(hi) T cells was associated with increased epithelial MLCK expression and myosin II regulatory light chain (MLC) phosphorylation as well as morphologic tight junction disruption. In vitro studies showed that TNF caused similar increases in MLCK expression and MLC phosphorylation, as well as barrier dysfunction, in Caco-2 monolayers only after interferon (IFN)-gamma pretreatment. This reductionist model was therefore used to determine the molecular mechanism by which IFN-gamma and TNF synergize to cause intestinal epithelial barrier loss. IFN-gamma priming increased TNFR1 and TNFR2 expression, and blocking antibody studies showed that TNFR2, but not TNFR1, was required for TNF-induced barrier dysfunction. Transgenic TNFR2, but not TNFR1, expression allowed IFN-gamma-independent TNF responses.

CONCLUSIONS:

IFN-gamma primes intestinal epithelia to respond to TNF by inducing TNFR2 expression, which in turn mediates TNF-induced MLCK-dependent barrier dysfunction. The data further suggest that epithelial TNFR2 blockade may be a novel approach to restore barrier function in intestinal disease.

IFN -γ primes Caco-2 monolayers for TNF-induced barrier dysfunction.A. Caco-2 cells were pre-incubated with IFN-γ, 10 ng/ml, for 24 hours prior to transfer to fresh media, without IFN-γ, either with or without TNF, 2.5 ng/ml, as indicated. Monolayers were then harvested and analyzed by SDS-PAGE immunoblot for MLCK expression, MLC phosphorylation, and total MLC as a loading control. When IFN-γ-primed cells were treated with TNF, MLCK expression increased 2.1±0.1-fold and MLC phosphorylation increased 3.0±0.6-fold (p < 0.05 for both). Other treatments did not significantly alter MLCK expression or MLC phosphorylation. B. TER of control monolayers (black triangles) was measured for 8 hours after transfer to fresh media. Addition of TNF, 2.5 ng/ml, to the basal media had no effect on TER (black circles). Pre-incubation with IFN-γ, 10 ng/ml, for 24 hours prior to transfer to fresh media without IFN-γ, also had no effect on TER (white triangles). In contrast, the TER of monolayers pre-incubated with IFN-γ prior dropped within 3 hours after transfer to fresh media with TNF (white circles, p<0.01). Data are mean ± SE of triplicate monolayers. Data are representative of more than 10 independent experiments. C. All monolayers were pre-incubated with IFN-γ, 10 ng/ml, for 24 hours. Transfer to fresh media without cytokines (white bar) had no effect on TER, while transfer to media with 2.5 ng/ml TNF in the basal chamber induced marked TER loss (black bar, p<0.02). Transfer to media with 2.5 ng/ml TNF in the apical chamber had no effect on TER. Data are mean ± SE of triplicate monolayers 8 hours after transfer and were normalized to the overall mean TER of monolayers pre-incubated with IFN-γ. Data are representative of 4 independent experiments.

IFN-γ upregulates both TNFR1 and TNFR2 in Caco-2 monolayers.A. RT-PCR and SDS-PAGE immunoblot analyses show that both TNFR1 and TNFR2 mRNA and protein expression are upregulated after 24 hours of culture with IFN-γ added to the basal chamber (p<0.01 for both). Data are representative of 4 independent experiments, each with duplicate samples. B. Immunofluorescence of monolayers before or after 24 hours of culture with IFN-γ confirms increased surface expression of TNFR1 and TNFR2. The images shown were taken at focal planes basal to the tight junction and TNF receptors were only detected on lateral membranes. Exposures and post-processing were identical for images of each TNF receptor. Bar = 10 μm.

Transgenic TNFR1 expression does not confer TER responses to TNF in the absence of IFN-γ-priming.A. SDS-PAGE immunoblot of TNFR1 comparing two independently-generated TNFR1-transfected clones and nontransfected control and IFN-γ-primed Caco-2 monolayers. Data are representative of 3 independent experiments.B. Treatment of TNFR1-transfected monolayers with TNF at doses from 0.1 ng/ml to 10 ng/ml does not induce significant TER loss. Data are mean ± SE of triplicate monolayers normalized to TER of monolayers not treated with TNF. Data are representative of 3 independent experiments.C. Although TNFR1-transfected monolayers are not responsive to TNF without IFN-γ, they are able to regulate TER in response to TNF, 2.5 ng/ml, after IFN-γ-priming (p<0.02). Data are mean ± SE of triplicate monolayers 8 hours after transfer to TNF. Data were normalized to TER prior to TNF treatments and are representative of 4 independent experiments.